Summary: Serological studies confirmed that inlfuenza B viruses have undergone marked antigenic changes from the previous vaccine strain. The data were used by the USPHS and WHO to recommend inclusion of a new strain, a B/Sichuan/379/99-like virus,in inactivated influenza vaccines for the 2001-2002 season. Since the named strain did not replicate well enough to be of practical use in vaccine production, three other strains (B/Johannesburg/5/99, B/Victoria/504/2000, and B/Guangdong/120/2000) werre identified that could be used for vaccine production. Our laboratory produced reference reagents for the B/Victoria/504/2000 and the B/Guangdong/120/2000 strains, which manufacturers helped to identify as the most useful ones. The reference reagents facilitated qualification and release of approximately 75 million doses of influenza vaccine for the United States. In addition, as reference for production purposes, we cloned a new influenza B virus strain (B/Johannesburg/5/99 RESVIR 18) with increased ability to replicate in eggs; this strain was distributed to WHO Influenza Centers, national laboratories and manufacturers for evaluation. Although the strain was not selected for use in vaccine production, it represents an initial attempt to produce high yield influenza B viruses to facilitate increased vaccine supplies for the United States. In response to the appearance of avian influenza A virus subtypes in man and the continued circulation of H5, H9, and H6 influenza A hemagglutinin subtypes in poultry in Hong Kong and China, we have continued preparations for the possibility of a pandemic. We have produced reference antisera against H5 and H9 influenza subtype hemagglutinins, and anticipate producing additional reference antisera for other subtype hemagglutinins as we are able to obtain and prepare materials. In order to prevent epidemic/pandemic influenza, high growth vaccine candidates are necessary for efficient preparation of influenza virus vaccines. Most wild type viruses with appropriate antigenic characteristics do not grow to sufficient high titer in eggs to support vaccine production. Our research program addresses several critical and unaddressed research needs of the influenza vaccine program to ensure sufficient vaccine supply by understanding molecular mechanisms of influenza virus replication and enhance production of influenza vaccine. The matrix gene is one of the "internal" genes that plays an important role in viral replication by interaction with ribonucleoproteins (RNP) and with membrane. Our previous studies demonstrate that matrix gene product, M1 protein, modulates viral assembly by enhancing formation of and interactions with RNP. Interaction of M1 with RNP facilitates translocation of RNP from nucleus to cytoplasm and may enhance viral replication. Association of M1/RNP complexes with cell membrane and membrane proteins such as HA, NA may relate to a role for M1 in the budding of newly synthesized virions from the cell surface. In addition to further examination of the functional domains of M1 on RNP formation and nuclear export, which are the important stages during viral replication, we studied association of M1 with cell membrane by characterization of virus-like particle formation from the plasmids coding for M1 gene in cells. Our preliminary studies suggests that M1 affects virus budding by interaction with cellular membrane and membrane proteins.